SEM/EDX analysis of stomach contents of a sea slug snacking on a polluted seafloor reveal microplastics as a component of its diet

Understanding the impacts of microplastics on living organisms in aquatic habitats is one of the hottest research topics worldwide. Despite increased attention, investigating microplastics in underwater environments remains a problematic task, due to the ubiquitous occurrence of microplastic, its multiple modes of interactions with the biota, and to the diversity of the synthetic organic polymers composing microplastics in the field. Several studies on microplastics focused on marine invertebrates, but to date, the benthic sea slugs (Mollusca, Gastropoda, Heterobranchia) were not yet investigated. Sea slugs are known to live on the organisms on which they feed on or to snack while gliding over the sea floor, but also as users of exogenous molecules or materials not only for nutrition. Therefore, they may represent a potential biological model to explore new modes of transformation and/or management of plastic, so far considered to be a non-biodegradable polymer. In this study we analysed the stomachal content of Bursatella leachii, an aplysiid heterobranch living in the Mar Piccolo, a highly polluted coastal basin near Taranto, in the northern part of the Ionian Sea. Microplastics were found in the stomachs of all the six sampled specimens, and SEM/EDX analyses were carried out to characterize the plastic debris. The SEM images and EDX spectra gathered here should be regarded as a baseline reference database for future investigations on marine Heterobranchia and their interactions with microplastics.

www.nature.com/scientificreports/ spectra obtained from environmental samples that is 'non-virgin microplastics' . These data will help to deliver a reference baseline of microplastic contamination for future monitoring studies, not only in areas under severe contamination levels as in the Mar Piccolo of Taranto, but also in less polluted areas under chronical, long-time exposure.

Materials and methods
The sampling locality was in the Mar Piccolo (40° 48′ N, 17° 25′ E) in Taranto (Apulian, Ionian Sea, Central Mediterranean Sea), a semi closed highly polluted basin, hosting a high number of industries and intensive farming of mussels and fish (Fig. 1). Six specimens of Bursatella leachii (Fig. 2) were collected by scuba diving at 10 m depth (Fig. 1a,b) and identified according to the external morphological diagnostic characters 87,88 . www.nature.com/scientificreports/ Each sample was observed in situ and in laboratory, photographed using a stereomicroscope and a microscope, preserved in 95% ethanol for future analysis and deposited as voucher at the Department of Science of the Roma Tre University (Rome, Italy). To reduce possible contamination of samples, with the consequent overestimation of microplastic detected, preventive measures were applied. In particular, specimens were manipulated underwater without using gloves, wrapped in aluminium foil before having been placed in a tank and finally transferred to the laboratory where they were suddenly stored in alcohol and analysed. Furthermore, in each step of the laboratory analyses, only glass materials washed with micro-filtered water were used.
Anatomical dissection. Analyses of the internal anatomy of the collected specimens were carried out by anatomical dissection under the stereo microscope at different magnification levels. The digestive system, from the oesophagus (taken right at the end of the gizzard) to the terminal anus, was isolated from the rest of the body and prepared for the next microplastic extraction protocol. Stomachal content was observed at the stereomicroscope and the ingested particles that were undoubtedly not plastics were separated for further detailed observations. The rest of the stomachal content, including visible fibres and microplastic debris, was placed in a separate 50 ml tube.

Microplastic extraction and samples preparation.
Prior to carrying out the microplastics extraction and characterization, the digestive system of each specimen was rinsed with pre-filtered (0.22 μm) deionized water and centrifuged to eliminate alcohol used to store them. Subsequently each pellet was incubated with 10% of KOH (w/v) solution prepared using KOH pellets (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and double-distilled water. Then they were placed on an agitation plate (IKA RT15, Staufen, Germany) set at 300 rpm and 60 ± 1 °C for 24 h. After digestion, all samples were filtered on 90 mm diameter GF/C glass microfibre filters (Whatman, Velizy-Villacoublay, France) using a vacuum system. Filters were then placed in closed Petri dishes until subsequent analysis. For the first characterisation, filters were observed under a stereomicroscope (Nikon SMZ25, Tokyo, Japan), allowing the identification of plastic particles. Items with characteristics similar to plastic polymers were characterized by size (< 100 µm; 100-500 µm; ˃500 µm) and colour. Filtered fragments and fibres were then rinsed in distilled water and mounted on double-sided adhesive carbon tabs on aluminium SEM stubs for successive SEM/EDX analyses. SEM/EDX analyses. SEM/EDX analysis was conducted on individual candidate microplastics selected by optical microscopy from the glass microfibre filters through which the treated B. leachii guts were filtered. SEM/ EDX allowed many potential microplastic particles to be screened in a relatively short time. SEM/EDX screening utilized surface morphology and elemental composition to determine whether each particle was potentially a plastic. The analyses were conducted using two different microscopes: The JSM-6480LV Scanning Electron Microscope (JEOL Ltd., Tokyo, Japan) with a Sirius SD Energy Dispersive X-ray Spectometer (iXRF Systems Inc., Houston, USA) (hereafter as SEM-JEOL) and the Sigma 300 VP Field Emission Scanning Electron Microscope (ZEISS, Oberkochen, Germany) (hereafter as FESEM-ZEISS). The former was used to the preliminary morphological assessment and to carry out the detailed microanalysis while the latter to obtain high resolution images useful to the in-depth observations of the morphological details. After checking that there was no sample charging under the electron beam, to avoid the contamination due to chemical artifacts introduced by the metal coating of the samples, these latter were initially analysed without the gold-coating step. The SEM-JEOL provided low resolution imaging of particle surface structures (not shown), as well as elemental composition signatures. Spectra of the chemical composition of the debris analysed were then compared with those already present in literature and related not only to microplastics but also to other organic compounds that cannot be removed by treatment with the KOH solution (like for example cellulose and chitin) but that could anyway be ingested by B. leachii.
To obtain high resolution SEM images, fragments and fibres previously mounted on the aluminum stubs and analysed with EDX, were afterwards gold coated in an Emitech K550x sputter unit, and finally examined by FESEM-ZEISS up to × 5000 magnification. The integration between results obtained by SEM and EDX methods and taking into consideration that samples were previously digested with 10% of KOH (w/v) solution which www.nature.com/scientificreports/ means that a lot of organic compounds were consequently excluded from the dataset under investigation, allows establishing if the analysed samples were effectively plastics or not.
Compliance with ethical standards. All applicable international, national and/or institutional guidelines for sampling, care and experimental use of organisms for the study have been followed and all necessary approvals have been obtained.

Results
Digestive systems from six individuals of B. leachii (Fig. 2a,b) from the Mar Piccolo of Taranto (Fig. 1b,c) were dissected and isolated ( Fig. 3) for preliminary microscopic analyses. Microplastics were found in the stomach contents of all specimens (Fig. 4) and sorted according to their morphologies into fragments and fibres and by size and colour. Fibre particles were fewer than the number of irregular fragments in five out of six analysed stomach contents (Table 1).

Microplastic extraction and analyses.
In the gastrointestinal tract of six B. leachii individuals, many microplastics were detected, visible onto the glass microfilters ( Fig. 4a-d). The number and type of microplastics detected in each analysed sample were reported in Table 1. The microplastics size distribution is presented in Fig. 4b. The size class distribution revealed a marked prevalence for particles smaller than 100 µm (54%), followed by particles with a range 100-500 µm (24%) and particles over 500 µm (22%). A total of 11 chromatic components were observed. The black colour dominated with 45.60% of particles found in specimens (Fig. 4a).
Other colours representing important proportions were transparent (4.75%), white (11.87%), brown (10.67%), orange (9.92%), blue (6.65%), bi-colour (5.46%), green (4.27%) and small percentage of the other colours. In addition, we reported the size distribution for each colour, as shown in Fig. 4c. The particle sizes varied for every colour, without a predominance of size in the case of the most common colours. Only the small percentage of SEM/EDX analyses. Surface texture created by environmental exposure is one of the primary characteristics that can be used to screen for microplastics by electron microscopy. Morphological analysis of the microplastic particle surfaces often revealed degradation and abrasion signs, suggesting mechanical weathering processes 89,90 , which were observed in this study. SEM/EDX analyses provided high resolution pictures of the particles surface structure of the fibres and fragments analysed, as well as their elemental composition signatures. This information was used to screen for likely microplastics and rule out non-plastics. Since it is known from literature that the most common kind of plastics, as Polypropylene (PP) and Polyethylene (PE), show a strong Carbon EDX peak 90 , and considering that we are dealing with plastics extracted from an open environment, possibly affected by all the variety of existing plastics, we searched for spectra showing a significant concentration of Carbon to be the possible candidates for microplastics. Resultant spectra were compared with some reported by published studies carried out in laboratory and using previously known plastics polymers that were used as reference. Examples of spectra from canonical microplastic fragments and fibres are shown in Fig. 5 as well as spectra from different kind of plastic (Fig. 6) where it is easy to see the Carbon peak (C) and other few additional elements characteristic of other plastic types. Spectra obtained were characterized by a high variability that perfectly reflects the huge variety of plastic currently available from natural environment. SEM/EDX analysis from fragments and fibres which were determined to be non-plastic (such as natural fibres, mollusc's shell fragments and debris of plant organisms) were also shown (Fig. 7).

Discussion
Morphological and elemental composition analyses of the stomachal content of Bursatella leachii from a highly polluted environment revealed the presence of microplastic fibres and fragments in all the studied individuals. Despite increased international attention, investigating microplastic in environmental samples is a difficult task, because of its wide range of possible interactions with the living biota and because microplastic includes different organic polymers which can be chemically and mechanically modified by environmental factors like the weather, hydrodynamic forces, solar radiation, the presence of other contaminants, the occurrence of biofouling, etc. [91][92][93][94][95][96] . The complexity of the processes of cause/effect characterizing microplastic implies the use of different protocols each of them optimized for a specific target of study. Additionally, even if studying microplastic is nowadays of a central importance and several standardized protocols of extraction and analysis have been published [97][98][99][100][101][102] , the continuous search for the most effective or performing one is still ongoing [103][104][105] . Furthermore, even though considerable research effort focused on several target species, (mainly vertebrates from fish to mammals) 17,19,21 , few invertebrates were investigated so far, and, among them, filter-feeder molluscs (bivalves) were mainly studied 15,31,39,57 . To date, interactions between environmental microplastics and Heterobranchia remained neglected, perhaps due to the difficulties of studying small and infrequent animals that are characterized by soft and very delicate internal anatomy; however, bridging the knowledge gap on these benthic consumers-known to unceasingly explore soft and hard bottoms on the seafloor in search of food and with different trophic preferences-is indeed a highly promising challenge. In fact, the potential of marine Heterobranchia is high since these gastropods are characterized by unique defensive strategies like the ability to accumulate and, in most of the cases, modify, chemical active compounds obtained from the diet. Considering that microplastic is everlasting due to the absence of known multicellular organisms able to digest it, the capacity showed by marine Heterobranchia to modify foreign chemical molecules may be of great interest under a potential biotechnological perspective.
In this framework, we investigated stomachal content from Bursatella leachii, an Aplysiidae living in Mar Piccolo of Taranto (Ionian Sea, Mediterranean Sea), a coastal aquatic environment under high anthropogenic pressures, particularly exposed to plastic pollution (Fig. 1). The combination of the high-resolution SEM morphological observations with the EDX elemental composition of the debris was very useful to investigate and identify microplastics in the digestive trait of B. leachii. In fact, considering that microplastic can bind with other pollutants already present in the environment and taking into consideration that Mar Piccolo of Taranto host several anthropogenic pollutants, it can be hypothesised that the collected microplastic may show an atypical chemical composition, reflected by a non-canonical spectrum. This characteristic would eventually affect results from other kind of techniques like for example the FTIR analyses, since it is based on the perfect match between the spectra investigated and the canonical spectra of plastic already available in the reference public libraries. Being extracted from individuals living in a natural environment and not from 'in laboratory study' , EDX spectra obtained were characterized by a high variability that perfectly reflects the vast diversity of plastic polymers now recorded in natural environments 48 .
The data presented here can be considered as preliminary, as they are based on dissection and analysis of six specimens only: a larger sampling will be required to corroborate and enlarge the value of these initial observations. However, the information gathered so far seems to be indicative of a consistent pattern. Firstly, fibres and www.nature.com/scientificreports/ small fragment particles were abundant in all the Bursatella stomachs analysed. This could be due to a selection made by the individuals with the exclusion of larger fragments and in favour of smaller ones or fibres stuck to the algae they feed on. Anyway, the dominance of fragments instead of fibre particles is of outmost interest and unexpected, since almost all previous studies on benthic animals report the opposite condition. This finding may corroborate the hypothesis of an active choice made by B. leachii, ingesting size-and shape-specific plastic debris and not just the most abundant ones, as usually happen for filter-feeders and/or non-selective detritivore organisms. In fact, fibres are expected to be copious in Mar Piccolo sediments, where they derive from abundant textile waste, degrading fishing lines, and particularly from the plastic nest nets widely used in mussel aquaculture (Fig. 1c-f), a major source of local contamination 24 . These preliminary data will represent baseline information for future comparative studies. Indeed, the analyses of high-res SEM images, together with the corresponding EDX spectra of the analysed fragments and fibres, highlighted the variability of the pool of debris that could be found in the field, with reference to already published information on compounds belonging to both natural and plastic materials. In fact, there are some natural materials that may resist the KOH digestion and therefore may require a future, in-depth analysis to distinguish them from the microplastic dataset and to avoid misidentifications. Regarding the microplastic debris found in B. leachii, the canonical spectra that could be identified as plastic are reported in Fig. 5 both related to fragments and fibres. These spectra are consistent with the ones from literature as microplastic 90,106 . Among these, an important class is that of fibreglass which includes different kind of plastic all of them characterized by Carbon (C), Oxygen (O), and Silicon (Si) 107,108 (https:// www. nrc. gov/ docs/ ML0530/ ML053 040493. pdf) where the glass fibres are commonly added to reinforce plastic structures 109,110 . This specific kind of plastic is part of the components detected in the present study (Fig. 6). Apart from the elements discussed till now, other interesting and unusual elements were found like Titanium (Ti), Barium (Ba) and Zinc (Zn) (Fig. 6). These elements are used as additives in some types of plastics thus driving a more precise identification 111,112 . SEM/EDX microanalysis from fragments and fibres which were found to be non-plastic were also shown to be useful reference for further in situ studies focused on animals in natural environments (Fig. 7). www.nature.com/scientificreports/ In fact, among the samples that were non-plastic, there was a natural cellulosic fibre (cotton) characterized by a typical twisted morphology and the presence of peaks resembling those reported for cellulose ( Fig. 7a-c) [113][114][115] . Also, we detected debris containing a high concentration of calcium carbonate (CaCO 3 ) (Fig. 7d-f), which is coherent with spectra reported for bivalves and gastropod shells 31,32,116,117 , tubes of sedentary polychaete worms 118 and therefore not associated with plastic. The almost equal concentration of Carbon and Oxygen (C and O) elements detected in a single fragment (Fig. 7g-i), together with the characteristic external morphology, is related to the cellulose and, indeed, to plant organisms and not to plastic. Another important result for consideration must be the absence of Nitrogen (N) in the EDX spectra of our samples. This element in fact is one of the main components of biological compounds (containing proteins and/or peptide bonds) such as egg capsules of the gastropod Rapana venosa (Valenciennes, 1846) 119 and eggshells of the Ascarididae nematode Ophidascaris baylisi Baylis (1921) 120 .
Investigating the variability among plastic materials is essential to fill the gap of knowledge still existing to date, avoiding misidentifications and errors in quantification of microplastic in the environments or into living organisms [91][92][93][94][95][96]121 . This last point is crucial and constitutes one of the main limits of the recent studies. In fact, most of them are carried out in laboratory and have used already known kinds of plastics which are therefore characterized by a known specific composition, while many other papers focused on plastic obtained by in situ studies but analysed using reference spectra from virgin plastic with known composition and eliminating those which are far from that reference. Anyway, the plastic debris found in a natural environment, and even more in a very polluted one, is not virgin plastic but instead a mixture of organic polymers plus a lot of different other elements that are added as additives or that are independently attracted by plastic due to chemical properties of the plastic itself. In fact, the microplastics found in the stomachs of B. leachii living in Mar Piccolo represent strong evidence of the high level of plastic contamination acting in this semi closed basin and the ease with which it enters the food chain as it is ingested by organisms that largely select the nutrients they eat. Interestingly, none of the stomachs analysed contained detritus, confirming that B. leachii is not a detritivore but selects plant organisms and a lot of other living organisms, in some way selecting them. We cannot exclude, therefore, that the microplastics were deposited on the surface of the prey ingested by the sea slugs. Anyway, it is interesting to note www.nature.com/scientificreports/ that once in the marine environment, microplastics are colonised by the 'plastisphere' as are collectively called the wide variety of microbial communities coating plastic debris and forming biofilms 121,122 . Considering that it was recently demonstrated the higher preference of some marine filter-feeders to eat microplastics coated with microbial biofilms instead of virgin microplastics, we can speculate the possible trophic preference of B. leachii for microplastics covered by cyanobacteria or other microbial biofilms. Anyway, this important ecological aspect is to date poorly known since most of the studies investigating the impacts of microplastics ingestion by aquatic organisms have currently used virgin plastic particles, which, however, do not reflect the real conditions of the sea 121 as the results here reported have also demonstrated. Another consideration can be made regarding the fact that plastic was found in all the specimens analysed independently if they had a lot of food in the digestive apparatus or not. This could be evidence of the possible persistence of plastic that would not be excreted with faeces. Taking into consideration that Heterobranchia have the capability to accumulate exogenous material and chemically modify it for defensive purposes, this observation becomes even more interesting. Further in-depth studies are needed to fulfil the gap of knowledge on this intriguing biological capability with potential highly innovative applications.
In conclusion, this work constitutes a baseline study useful for future in-depth investigations on the diet and faecal contents and for future comparisons with plastics contents from digestive traits of other taxa and with EDX spectra from other materials of difficult identification. Furthermore, it could be a reference database on the composition of locally originated plastics in the Mar Piccolo (due to its typical hydrology) that will be useful to simulate possible future scenarios on other polluted coastal areas and to predict the potential outcome of negative impacts on the biota.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information files). www.nature.com/scientificreports/